Disulfide Biotin Azide
Disulfide Biotin Azide is an azide-activated cleavable biotin probe that allows for efficient recovery of avidin-bound protein complexes in affinity-based assays. This cleavable probe is designed with disulfide bonds in the spacer arm. Under reducing conditions (50 mM dithiothreitol, 10 mM 2-mercaptoethanol or 1% sodium borohydride), the disulfide bonds are cleaved, releasing the biotin tag and any avidin conjugate bound to it.
Extraordinary strength of the streptavidin-biotin interaction allows for efficient capturing of even highly dilute targets; however, it makes recovery of proteins from affinity resins challenging. Conventional methods to elute biotinylated proteins from immobilized avidin include the following: (i) denaturation of streptavidin by boiling the resin in a denaturing buffer that may include high concentrations of chaotropic salts, (ii) trypsin digestion of proteins while they are bound to the resin, or (iii) elution of proteins with excess free biotin. These protocols can co-elute contaminant proteins by releasing nonspecifically bound proteins and/or naturally biotinylated proteins concurrently with labeled proteins. In addition, some of these methods can cause elution of high levels of resin-based peptides along with the proteins of interest, resulting in further sample contamination.
Disulfide Biotin Azide probe eliminates a major limitation of the streptavidin-biotin affinity purification. This reagent contains a biotin moiety linked to an azide moiety through a spacer arm containing a cleavable disulfide linker. Captured biomolecules can be efficiently released under mild conditions (50 mM dithiothreitol, 10 mM 2-mercaptoethanol or 1% sodium borohydride) and the small molecular fragment (188.25 Da) left on the labeled protein following cleavage. These features make the cleavable probe especially attractive for use in biomolecular labeling and proteomic studies.
- Szychowski, J., et al. (2010). Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition. J. Am. Chem. Soc., 132: 18351-60. [PubMed]
- Yang Y., et al. (2013). Cleavable Trifunctional Biotin Reagents for Protein Labeling, Capture, and Release. Chem. Commun., 48: 5366-86. [PubMed]